CN110495419A - A kind of mouse model of Huppert's disease extramedullary plasmacytoma - Google Patents
A kind of mouse model of Huppert's disease extramedullary plasmacytoma Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A61B17/34—Trocars; Puncturing needles
- A61B17/3468—Trocars; Puncturing needles for implanting or removing devices, e.g. prostheses, implants, seeds, wires
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
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Abstract
The invention discloses a kind of mouse models of Huppert's disease extramedullary plasmacytoma.The present invention is by the marrow tumor tissues of in vitro multiple myeloma patients (solid tumor organized outside Huppert's disease infiltration to marrow and marrow), it is transplanted to severe immune deficiency mouse after taking-up by puncture needle, it can tumor formation without human embryos bone, it is easy to operate simple and easy, the heterogeneity of bone marrow microenvironment and tumour is remained with, large-scale drug screening and pre-clinical assessment are suitable for.
Description
Technical field
The present invention relates to biomedicine fields, and in particular to a kind of mouse mould of Huppert's disease extramedullary plasmacytoma
Type.
Background technique
Huppert's disease (MultipleMyeloma, MM) is malignant tumour caused by a kind of plasma cell dyscrasias is proliferated, about
The 1%-2% for accounting for all malignant tumours accounts for the 10% of Malignancy.Multiple myeloma patients are mostly the elderly,
And with aging of population, the disease incidence of Huppert's disease is being risen year by year.The diagnosis of Huppert's disease is with marrow
Interior abnormal plasmocyte increases, and with the characteristics of occurring M albumen and multiple target organ damages in blood urine, clinical manifestation is hypercalcinemia, kidney
Insufficiency, anaemia, osteolytic lesion and outer marrow infiltration mass etc., the double characteristic with neoplastic hematologic disorder and entity tumor.
Huppert's disease appears in during progression of disease forms mass outside marrow, referred to as Huppert's disease marrow ectoplasm
Cytoma, also referred to as Huppert's disease outer marrow infiltration (Extramedullary Multiple Myeloma, EMM), are that MM is being sent out
During exhibition outside tumor cell invasion to marrow caused by the histoorgans such as muscle ligament, subcutaneous, intraspinal tube, chemotherapy is usually invalid,
Even occur in chemotherapy process, belongs to relapsed or stubborn myeloma.
There are many kinds of the chemotherapy therapeutic schemes of Huppert's disease, such as tradition MP scheme (melphalan and Po Ni
Pine), (cyclophosphamide is relied on and is moored by VMPC scheme (vincristine, melphalan, cyclophosphamide and methylprednisolone) and CEVAD
Glycosides, vincristine, Doxorubicin and dexamethasone) etc., in addition to there are also hematopoietic stem cell transplantation schemes and emerging for chemotherapy
Target therapeutic agent lenalidomide, bortezomib, Carfilzomib etc..The new drug and the new mould of new departure for treating Huppert's disease
Formula is also continuing to bring out, due to the height heterogeneity of Huppert's disease, and a variety of underlying diseases of Chang Bingfa, Huppert's disease
Resistance problems are prominent, therefore, how to evaluate chemotherapy of patients scheme and judge whether effective critically important, new drug development and various treatments
The assessment of method be unable to do without the animal model close to clinical practice.Mouse mould applied to the source of people of Huppert's disease at present
Type is few, the SCID-hu multiple myeloma models having been reported, and is to inject people's Huppert's disease using human embryos bone
Cell creates the humanized mouse model of bone marrow microenvironment, but since embryo's bone is not easy to obtain, this model is using less.Cause
This, the animal model of humanized's Huppert's disease is suddenly to be improved.
Source of people Tumor Xenograft Models PDX-model (Patient-DerivedXenograft Model) is by tumour
The fresh tumor tissue of patient is transplanted in severe immune deficiency Mice Body, is tumor model of the tumor proliferation to establish.PDX
Model remains the histopathology of Primary Tumor and the characteristic of growth and transfer.It still is able to utmostly after multiple passage
Reservation tumour-specific.It can be used for personalised drug to screen, the preclinical models of new drug development, treatment side after multidrug resistance
The improvement of case, PDX model can be used as the effective tool of assessment antineoplastic evaluating drug effect.
Summary of the invention
The object of the present invention is to provide a kind of mouse models of Huppert's disease extramedullary plasmacytoma.
The present invention provides a kind of construction method of source of people Huppert's disease extramedullary plasmacytoma mouse model, including as follows
Step: in vitro source of people marrow tumor tissue implantation immunodeficient mouse is subcutaneous, obtain the outer thick liquid cell of source of people Huppert's disease marrow
Tumor mouse model.
The implantation is implanted by puncture needle.
The immunodeficient mouse can be NOD/SCID mouse or NCG mouse or NSG mouse.
The volume of implanting tissue is 10mm3。
Implantation position is upper limb and the lower limb middle position on the subcutaneous right side of mouse.
When tumor tissues reach after implantation, 800-1200mm3When, model construction is completed.
The mouse model meets following feature:
(1) tumor tissues detect the morphological feature for retaining source of people marrow tumor tissue through pathology；
(2) tumor tissues are detected as CD38, CD138 positive through immunophenotype.
The source of people Huppert's disease extramedullary plasmacytoma mouse model that the method obtains can carry out later passages.
It is inoculated with the same generation mouse of same marrow tumor tissue, when a wherein mouse meets features described above through detection, remaining
Mouse can be used as mouse model for study or later passages.
The present invention also protects in vitro source of people marrow tumor tissue preparing the application in product, and the purposes of the product is preparation
Source of people Huppert's disease extramedullary plasmacytoma mouse model.
The present invention also protects a kind of Huppert's disease drug screening method, includes the following steps:
(a) mouse model is constructed according to the method for any description above；
(b) drug screening is carried out using the model of step (a) preparation.
The in vitro source of people marrow tumor tissue of any description above is the in vitro Huppert's disease leaching from tumor patient
Moisten the solid tumor organized to outside marrow and marrow.
By the marrow tumor tissue of in vitro multiple myeloma patients, (Huppert's disease is infiltrated to marrow and marrow the present invention
The solid tumor of outer tissue), it is transplanted to severe immune deficiency mouse after taking-up by puncture needle, is without human embryos bone
Can tumor formation, it is easy to operate simple and easy, remain with the heterogeneity of bone marrow microenvironment and tumour, be suitable for large-scale drug screening and
Pre-clinical assessment.
Detailed description of the invention
Fig. 1 is tumor growth curve.
Fig. 2 is model mice.
Fig. 3 is tumor tissues.
Fig. 4 is histopathology result.
Fig. 5 is histogenic immunity Phenotypic examination result.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
NOD/SCID (non-obese diabetes/severe combined immunodeficiency) mouse, female, 5 week old: dimension company, tonneau China.
Specific step is as follows for histopathology in embodiment:
(1) 5mm × 5mm × 2mm tissue is taken, is put into 4% paraformaldehyde after PBS washing and fixes 12h, carry out paraffin packet
It buries, and is sliced.
(2) slide cut 30min in 70 DEG C of baking ovens is taken, deparaffnize is removed, then successively carries out dimethylbenzene I dewaxing 10min,
Dimethylbenzene II dewaxing 10min, then puts 100%, 95%, 90%, 80%, 70% ethanol dehydration 5min.
(3) PBS is washed 3 times, and slice is put into hematoxylin and dyes 20min, and tap water rinses 15min, and slice is put into 1%
It fades in ethanol solution of sulfuric acid, 2-10s.
(4) tap water rinses 15min, is sliced into 50% ethyl alcohol, 70% ethyl alcohol, and each 5min in 80% ethyl alcohol is added dropwise 0.5%
Slice is put into 95% ethyl alcohol and washes away extra red, is then placed in anhydrous second by Yihong ethanol solution counterstain 1-3min
3-5min in alcohol sucks extra ethyl alcohol.
(5) slice is put into each 5min in dimethylbenzene I, II, neutral gum mounting, microscopic observation.
Specific step is as follows for histogenic immunity Phenotypic examination in embodiment:
(1) take tissue, with source of people tumor tissues treatment kits (HumanTumorDissociationKit,
MiltenyiBiotec) processing obtains tumor cell suspension.
(2) cell suspension is taken to count 1 × 10^7 cell, PBS washing, supernatant is abandoned in centrifugation, is resuspended in 100 μ l containing 5%
It in the PBS solution of FBS, is added streaming antibody FITC-CD38, APC-CD19, PE-CD138 (biolenged), control group is set
(antibody is not added to be dyed) is protected from light in 2-4 DEG C of incubation 30min.
(3) supernatant, PBS washing of the 1ml containing 5%FBS are abandoned in centrifugation, and centrifugation abandons supernatant, 300 PBSs of the μ l containing 5%FBS are added,
Flow cytomery phenotype.
The mouse model building of embodiment 1, Huppert's disease extramedullary plasmacytoma
1, the marrow tumor tissue of in vitro multiple myeloma patients (is organized outside Huppert's disease infiltration to marrow and marrow
Solid tumor) (be labeled as Patient) be stored in sterile RPMI1640 culture medium, be cut into size about 10mm3Cube
Fritter, using puncture needle be transplanted to NOD/SCID (non-obese diabetes/severe combined immunodeficiency) mouse it is subcutaneous (right side it is upper
Limb and lower limb middle position do not influence the activity of mouse four limbs).
2, it after completing step 1, takes a part to be used for histopathological examination in residue tissue, as a result sees Fig. 4's
Patient。
3, after completing step 1, a part is taken to carry out immunophenotype detection in residue tissue, the inspection result of patient is specially
CD38, CD138 are positive, and CD19 is negative.
4, after completing step 1, a part is taken to be placed in Liquid nitrogen storage in residue tissue.
5, after completing step 1, the growth curve of tumour after record transplanting measures tumour long (a), wide (b), calculates knurl product
V=1/2 (a × b2).As shown in Figure 1.When tumor tissues volume reaches 1000mm3P0 is obtained when left and right for mouse model (Fig. 2), is taken
Tumor tissues (Fig. 3) out is stored in sterile RPMI1640 culture medium, is cut into size about 10mm3Cube fritter, according to
It is subcutaneous that method in step 1 is transplanted to mouse, is passed on to tumour and (successively obtains P1 for mouse model, P2 for mouse mould
Type ..., be passaged to 3-5 generation can be used for large scale experiment), while by remaining tumor tissues according to step 2 and 3 method carry out
Detection, residue tissue take a part to be placed in Liquid nitrogen storage.
P0 is shown in the MouseP0 of Fig. 4, pathology of the P1 for mouse tumor tissue for the pathological examination result of mouse tumor tissue
Learn the MouseP1 that inspection result is shown in Fig. 4.
P1 is shown in Fig. 5 for the immunophenotype testing result of mouse tumor tissue.
Mouse model obtained by the above method remains with the heterogeneity of bone marrow microenvironment and tumour, and is passaged to P5 generation still
Keep pathology morphological feature and immunophenotype (CD38, CD138 are positive, and CD19 is negative).
In the above method, it is inoculated with the same generation mouse of identical tissue, it only need to be to a progress pathological examination and immune table
Type detection, if detection meet condition (pathological examination retain marrow tumor tissue morphological feature, immunophenotype detect CD38,
CD138 is positive), remaining mouse is as animal pattern.
Above-mentioned experiment can also obtain same effect using NCG mouse or NSG mouse substitution NOD/SCID mouse.
Claims (9)
1. a kind of construction method of source of people Huppert's disease extramedullary plasmacytoma mouse model, including the following steps: will be in vitro
Source of people marrow tumor tissue implantation immunodeficient mouse is subcutaneous, obtains source of people Huppert's disease extramedullary plasmacytoma mouse model.
2. the method as described in claim 1, it is characterised in that: the implantation is implanted by puncture needle.
3. method according to claim 1 or 2, it is characterised in that: the immunodeficient mouse is NOD/SCID mouse or NCG
Mouse or NSG mouse.
4. method as described in claims 1 to 3, it is characterised in that: the volume of implanting tissue is 10mm3。
5. the method as described in Claims 1-4 is any, it is characterised in that: implantation position be the subcutaneous right side of mouse upper limb and
Lower limb middle position.
6. method as claimed in claim 1 to 5, it is characterised in that: when tumor tissues reach 800-1200mm after implantation3
When, model construction is completed.
7. the method as described in claim 1 to 6 is any, it is characterised in that: the mouse model meets following feature:
(1) tumor tissues detect the morphological feature for retaining source of people marrow tumor tissue through pathology；
(2) tumor tissues are detected as CD38, CD138 positive through immunophenotype.
8. in vitro source of people marrow tumor tissue is the preparation multiple marrow of source of people preparing the application in product, the purposes of the product
Tumor extramedullary plasmacytoma mouse model.
9. a kind of Huppert's disease drug screening method, includes the following steps:
(a) mouse model is constructed according to any method of claim 1 to 7；
(b) drug screening is carried out using the model of step (a) preparation.
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